Most commonly used techniques for analytical separations (such as for example gas chromatography, periodically referred to herein as “GC”; liquid chromatography, periodically referred to herein as “LC”; and capillary electrophoresis, periodically referred to herein as “CE”; etc.) begin with the injection of a plug of a multi-component sample at the beginning of a separation column and proceed with the migration of the sample plug along the column. Separation occurs as each analyte in the sample migrates with a different speed and so can be individually detected at the end of the column. There are two primary drawbacks to this type of separation. First, the injected sample plug must be very narrow to achieve good resolution of the migrating analytes. Second, the widths of the individual analyte bands increase and their amplitudes decrease as they migrate along the column. Consequently, it can be difficult to achieve both high resolution and low concentration detection limits in the same analysis. For GC in particular, and to a lesser extent for LC, a number of methods have been developed for the injection of a large amount of sample to improve detection limits followed by compression of the sample plug into a narrow starting band required for high resolution. There are significant disadvantages and limitations to these methods however. The most common problems are thermal degradation of analytes due to the high temperature of the injection chamber and biasing of either high or low affinity analytes.
A different approach to analytical separations is the use of an equilibrium gradient focusing method such as isoelectric focusing (IEF). With this type of approach, a gradient (in pH, electric field, density, temperature, etc.) is formed along the length of the separation column and different analytes are separated as they are made to focus or accumulate at different positions along the gradient. A major advantage of this strategy, referred to as “analyte focusing,” is that it combines the concentration and separation of analytes into one step. As a result, peaks become narrower and more concentrated as the separation progresses. A second important advantage is that the width of the injected sample plug need not be carefully defined, since the analytes will move toward and be focused at their respective positions regardless of how they were introduced into the column. Consequently, it is relatively easy to achieve both high resolution and low detection limits.
To achieve analyte focusing at a stationary position requires either a single force that changes direction at the focusing position or a combination of two counteracting forces arranged so that the sum of the forces changes direction at the focusing position. However, in LC and GC, there is only one force, the flow of mobile phase through the column. The flow cannot be made to change direction in the middle of the column. And so, it appears that it would not be possible to achieve the advantages of a focusing mode separation in LC or GC.
Regardless, it would be desirable to provide a method and related system for performing chromatography in an equilibrium gradient focusing mode. More specifically, it would be desirable to provide a technique and related system whereby analyte focusing could be performed in a chromatography process such as gas chromatography or liquid chromatography.
Attempts have been made by prior artisans to perform a chromatographic process based upon an equilibrium gradient focusing mode as opposed to a time-based migration mode, which nearly all conventional chromatography processes employ. In the 1950s, as GC was being developed, an equilibrium gradient focusing mode of GC was investigated—primarily by a Russian group led by Zhukhovitskii and Turkel'taub. Their results demonstrated the validity of the idea but were limited ultimately by the slow thermal equilibrium of the large-bore GC columns used at the time. The advent of microbore GC (and LC) columns in the 1970s would seem to have been an opportunity to try the idea again, but by then most research groups seem to have been wedded to the conventional approaches of isothermal and temperature-programmed (with a temperature gradient in time only) chromatography. It was not until the 1990s that the approach was tried with capillary columns. Even then, as far as is known, it was only pursued by two groups, and their focus was primarily on using the technique to increase the peak capacity per unit time of GC separations. Specifically, a group led by Wayne A. Rubey investigated the use of both spatial and temporal temperature gradients in capillary GC applications. Descriptions of Rubey's work in this regard are noted in “An Instrumentation Assembly for Studying Gradient Programmed Gas Chromatography,” Rev. Sci. Instrum., 65 (9), September 1994, p. 2802-2807; U.S. Pat. No. 4,923,486 to Rubey; and International Patent Publication No. WO 2006/127490 to Rubey et al. Another group, Phillips and Jain published two papers on this subject in 1995, “On-Column Temperature Programming in Gas-Chromatography Using Temperature-Gradients Along the Capillary Column,” Journal of Chromatographic Science, 33 (10), October 1995, p. 541-550; and “High-Speed Gas-Chromatography Using Simultaneous Temperature-Gradients In Both Time And Distance Along Narrow-Bore Capillary Columns,” Journal of Chromatographic Science, 33 (11), November 1995, p. 601-605.
The conclusions stated in the work by Phillips and Jain were disputed by a leading researcher in the field. In 1997, LM Blumberg published “Focusing Cannot Enhance Resolution or Speed Limit of a GC Column,” J. Chromatographic Science, Vol. 35, September 1997, p. 451-454. In that letter, Blumberg disputed the previous claims of Phillips and Jain, and also the previous work by Rubey. After it was theoretically proven that the earlier claims by these two groups were essentially impossible, publication on the subject essentially stopped.